The present invention relates, in general to the use of antioxidant compounds, also referred herein as antioxidants, for the treatment of ischemic head injuries. More particularly, the present invention relates to novel brain targeted low molecular weight, hydrophobic antioxidants and their use in treatment of ischemic head injuries, such as, but not limited to stroke and head trauma.
Correlation Between Oxidative Stress and Various Neurodegenerative Pathologies
In the last few years evidences have accumulated which connect oxidative stress (OS) with the pathogenesis of Parkinson""s, Alzheimer""s Creutzfeldt-Jakob""s diseases and other human neurodegenerative disorders (Olanow, 1990, 1993; Fahn and Cohen, 1992; Cafe et al., 1996, Brown et al., 1996; Thomas et al., 1996).
These studies were initiated (i) since outo-oxidation of levodopa and dopamine is known to produce oxygen free radicals, H2O2, quinones and semiquinones, the later two are high molecular weight polymers possessing an aromatic structure and are therefore potentially toxic and (ii) since post-mortem studies in Parkinson""s disease patients showed a dramatic decline in the levels of endogenous reduced glutathione (GSH), which is, as is further delineated hereinbelow, essential for maintaining the oxidative state of the cells. The decrease in reduced glutathione levels progresses from the pre symptomatic Parkinson""s disease condition to the advanced clinical Parkinson""s disease condition.
Two possible explanations may account for the role played by oxidative stress in the pathogenesis of Parkinson""s disease.
According to a first hypothesis it is assumed that cells of the substantia nigra (SN), are constantly under oxidative stress due to the oxidation of the catechol ring of dopamine. Dopamine, like other catecholamines, undergoes spontaneous oxidation to form semiquinones, oxygen free-radicals and H2O2 as metabolic by-products. In addition, one of the disposal routs for dopamine is its enzymatic oxidation by MAO (monamine oxidase) type A or B which, like the spontaneous oxidation, creates semiquinones, oxygen free radicals and H2O2.
These products may cause accumulative oxidation damage within the substantia nigra cells, and eventually lead to cell death. Non-affected cells increase the turnover of dopamine, which in turn, generates more toxic free radicals. Indeed, it was shown that in the presence of H2O2 and copper ions, dopamine as well as L-dopa (levodopa) cause oxidative damage to DNA (Jenner 1994; Spencer et al., 1994).
According to another hypothesis it is assumed that Parkinson""s disease is caused by a substance of an unknown composition, similar to the toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which is enzymatically converted to the toxic metabolite 1-methyl-4-phenyl pyridine (MPP+) by MAO type B. Since this reaction releases free oxygen radicals, thereby increasing the oxidative stress imposed on the cells, similar mechanisms might affect the nigral cells in Parkinson""s disease. If this type of a process indeed occurs in Parkinsonian brains, than again, it would create a high level of oxidative stress in the dopaminergic cells at the substantia nigra.
The following findings demonstrate a strong indication for oxidative stress as a possible cause for the pathogenesis of Parkinson""s disease.
First, the iron content in the substentia Nigra of Parkinson""s disease patients was found to be significantly higher while ferritin content, the protein which bind free iron ions within tissues, was found to be significantly lower then normal values (Olanow 1990; 1993 and Jenner 1994). These two phenomena indicate a situation where the amount of free iron which acts as a catalyst in oxidation reactions, is abnormally high and thus may contribute to the speed of oxidation reactions at the substantia nigra of Parkinson""s disease patients. The above indication was given support when it was shown that injection of free iron directly into the substantia nigra of rodents caused the appearance of Parkinsonian symptoms, which symptoms could be overcome by addition of transferrin, the protein which binds free iron in the blood plasma (Jenner 1994).
Second, one of the protective mechanisms against oxidation processes in the brain is reduced glutathione (GSH), which upon oxidation to oxidized glutathione (GS), acts as a reducing agent. In the substantia nigra of Parkinson""s disease patients the level of reduced glutathione is significantly low, whereas the level of oxidized glutathione remains normal. Hence the oxidation potential in the substantia nigra of Parkinson""s disease patients is low. This change in the level of reduced glutathione is in all likelihood, specific for Parkinson""s disease. The oxidation products which are formed during spontaneous and enzymatic oxidation of dopamine, as described hereinabove, lower the level of reduced glutathione, and thereby increase the ratio of oxidized/reduced glutathione. Since the level of xcex3-glutamyl-cysteine synthetase, the enzyme which is a rate limiting enzyme in the biochemical pathway of glutathione synthesis, is normal, the ratio stays high and induces a state of oxidative stress in the cells. It is interesting that the level of the enzyme which is responsible for the removal of oxidized glutathione, xcex3-glutamyl transpeptidase in the cells is higher, as if the cells attempt to overcome the increased oxidative stress by trying to get rid of oxidized glutathione (Sian et al., 1994).
Third, additional evidence for the abnormally high oxidative stress in the Parkinsonian brain comes from a study of lipid oxidation products in the substantia nigra of Parkinson""s disease patients. In general, the level of unsaturated fatty acids is low in the substantia nigra, however, the level of lipid hydroperoxides which are the oxidation products of unsaturated fatty acids is high in the substantia nigra of Parkinson""s patients. This finding indicates the presence of an abnormally higher frequency of oxidation processes in the Parkinsonian substantia nigra (Jenner, 1994).
And finally, one of the animal models which are currently used for the study of Parkinson""s disease is created by the injection of 6-hydroxy-dopamine. Like dopamine, this false neurotransmitter elevates the level of the oxidation products during its degradation, thus leading to cell death. Since the biological half life of 6-hydroxy-dopamine is much longer and since it is readily taken-up by the cells, it increases the rate by which the animal develops the symptoms of the disease.
The different pathological makers of various neurodegenerative diseases e.g., Lewy bodies, plaques, etc., indicate different causal factors in the initiation of these diseases. However, there is growing evidence that once initiated, the progression of a large number of neurodegenerative diseases, is quite similar. Although the characteristic symptoms are descriptive for each neurodegenerative disease, it appears that elevation of the oxidative state of the cells at specific regions in the brain is an important factor in the etiology of Parkinson""s disease, basal ganglia degenerative diseases, motoneuron diseases, Alzheimer""s and also the Creutzfeldt-Jakob""s disease.
An indication for a role played by oxidative stress in the pathogenesis of Alzheimer""s disease was found while in a recent study, the relationship between the xcex2-amyloid protein fragments and oxygen radical formation was tested in a system that is highly sensitive and responds to free oxygen radicals. This system utilizes the vasoactivity of the blood vessel which, in the presence of xcex2-amyloid, enhances the phenylephrine mediated contraction of the vessels. Pre treatment of the blood vessel with superoxide dismutase (SOD), an enzyme that scavenges free oxygen radicals, eliminated the effect of xcex2-amyloid, namely, there was no enhancement of vasoconstriction. Whereas, if SOD was added after treatment with xcex2-amyloid protein, the protective effect of the radical scavenger was abolished (Thomas et al., 1996). Recently, other studies have shown that oxidative stress and free radicals production are linked to xcex2-amyloid fragment which includes amino acids 25-35 and may contribute to neurodegenerative events associated with Alzheimer""s disease (Cafe et al., 1996).
Possible indications for a role played by oxidative stress in the pathogenesis of Scrapie, spongyform encephalopathy (BSE) and Creutzfeldt-Jakob""s diseases are listed hereinbelow.
In a recent study, it was demonstrated that the toxic effect of Scrapie requires the presence of microglia cells which respond to a prion protein fragment (PrP106-126) by increasing their oxygen radical production. Interestingly, all these effects were absolutely dependent on mice that express the prion protein PrPc (Brown et al., 1996). The contribution of progressive oxidative stress to the state of various diseases and to mechanism of cell death is further demonstrated in a study by P. Jenner in The Lancet (1994) 344, 796-798, which is incorporated by reference as if fully set forth herein.
New Therapeutic Aspects
The use of glial cell-derived neurotrophic factor (GDNF) was established as a potential stimulant for the increase of dopamine levels in midbrain of rhesus monkeys (Gash et al., 1996). This study which extends previous results obtained with rodents, is promising as a potential treatment for Parkinson""s disease. However, like any other protein, GDNF cannot cross the blood brain barrier. Therefore, it can not be taken orally or be injected systemically. The only possible mode of administration would thus be via an intracerebral injection which would constitute a main drawback for such a treatment.
Similarly, in other neurodegenerative diseases such as Alzheimer""s and Creutzfeldt-Jakob""s, where the theory of free oxygen radicals appears to play a major role, there is no major breakthrough in therapy.
To overcome high oxidative stress it would be beneficial to augment the reduced state of the cells at the central nervous system (CNS). One of the possible ways to do it is by increasing the level of reduced glutathione or other scavengers of free radicals and free oxygen in the brain.
In general, in order to lower oxidative stress levels, various antioxidants are being used. The most common are vitamin E and vitamin C. However, vitamin E was found to be ineffective at decreasing the oxidative stress at the substantia nigra (The Parkinson Study Group, 1993, Offen et al., 1996) since this compound, although capable of crossing the blood brain barrier, is trapped in the cell membrane and therefore does not reach the cytoplasm where its antioxidant properties are needed. Vitamin C does not cross the blood brain barrier and therefore, cannot be used effectively for neurodegenerative diseases of central origin.
Recently, a similar approach for reducing the levels of free oxygen, was taken for the treatment of asthma (Bundy et al., 1995). A reactive oxygen inhibitor was synthesized (2,4-diaminopyrrolo-[2,3-d]pyrimidines) and after a successful pharmaceutical bio-availability and toxicity tests was selected for clinical evaluation.
A somewhat different approach involves stimulating the production of endogenous antioxidants, especially reduced glutathione. To this end a drug known as Procysteine which boosts cellular production of glutathione by loading the cells with cysteine is under clinical trials these days by Free Radical Sciences Inc. (CA, US) to treat conditions of acute respiratory distress syndrome (ARDS) which includes overproduction of oxidants or reactive oxygen species by the immune system. Other conditions in which overproduction of oxidants is experienced include but are not limited to amyotrophic lateral sclerosis, atherosclerotic cardiovascular disease and multiple organ dysfunction. See, for example, Charles Craig, 1996.
PCT/US97/23997 teaches novel brain targeted low molecular weight, hydrophobic antioxidants and the use of such antioxidants in treatment of central nervous system neurodegenerative disorders such as Parkinson""s, Alzheimer""s and Creutzfeldt-Jakob""s diseases and in treatment of conditions of peripheral tissues such as acute respiratory distress syndrome, amyotrophic lateral sclerosis, atherosclerotic cardiovascular disease and multiple organ dysfunction, in which oxidants are overproduced. PCT/US97/23997, however, fails to teach the use of such antioxidants for treatment in cases of ischemic brain injuries.
Ischemic Brain Injury
Ischemic brain injury, due to, for example, stroke or head trauma, is a major cause of morbidity and mortality in adults but also of neurodevelopmental impairment and disability. The critical reduction of local cerebral blood flow and the resulting ischemia are associated with a catastrophic cascade of events that may affect and determine survival or death of neurons, the final territory of infracted tissue and the neurological outcome. Prominent among the biochemical features of ischemic injury is the loss of cellular ATP, resulting in increased intracellular Na+ and Ca2+, and decreased intracellular K+. These ionic imbalances, together with a breakdown in cellular defense systems following ischemia, can contribute to enhanced local oxidative stress with a net increase in potentially harmful reactive oxygen species (ROS). Subsequent damage to lipids, proteins, DNA and inactivation of key cellular enzymes ultimately lead to cell death. Such damages occur after transient brain ischemia, and in the penumbral region of infarcts caused by permanent ischemia.
Although the precise mechanisms of neuronal loss are yet unclear, it is now clear that both apoptosis and necrosis are significant models of cell death following ischemia. Naturally, immediate re-establishment of arterial blood supply and oxygenation by clot lysis, e.g., using tissue plasminogen activator (tPA) can be helpful and positively affect for neurological prognosis. However, such an approach is impractical and may be too late for most patients with ischemic stroke. Therefore, it seems no less important to try and protect as many neurons as possible, particularly in the penumbra region. The suggested destructive mechanisms associated with ischemia are initiated by activation of glutamate receptors resulting in elevated intracellular Ca2+ and ROS formation. Three major approaches have been investigated to counteract and prevent ischemia-induced brain damage: (i) interfering with the excitatory action of glutamate; (ii) preventing intracellular accumulation of Ca2+; and (iii) preventing the destructive actions of reactive oxygen species (ROS). Interference with glutamate action can be achieved by: (i) facilitating mechanisms that maintain membrane potentials; (ii) blocking glutamate receptors; and (iii) inhibiting glutamate synthesis. Prevention of intracellular Ca2+ accumulation may be achieved by: (i) blocking Ca2+ channels; and (ii) facilitating endogenous Ca2+ homeostatic mechanisms. Destructive actions of ROS can be minimized by: (i) administration of ROS-scavenging drugs; (ii) upregulating endogenous ROS-scavenging mechanisms; and (iii) preventing leukocyte invasion of the affected brain tissue.
Most, if not all, of the currently available antioxidants cannot penetrate the blood brain barrier (BBB) from the systemic circulation and therefore fail to decrease the ischemic damage in brains. Several studies have shown that raising the endogenous antioxidant defense can rescue neurons during ischemia. For instance, it was shown that transgenic mice overexpressing bcl-2 (an anti-apoptotic, anti-oxidant gene) in their neurons, are more resistant to induced ischemia (Martinou et al., 1994).
Furthermore, overexpression of thioredoxin, a redox-active disulfide/dithiol in transgenic mice, attenuates focal ischemic brain damage (Takagi et al., 1999).
These studies and others, strongly suggest that antioxidants that would effectively cross the BBB and penetrate into the ischemic tissue mainly at its penumbra, may help to maintain the redox status of the neurons, decrease ROS-associated neuronal damage. Thus reduce the neurological impairment and disability. If successful, the clinical, social and economical impact of the proposed novel strategy is enormous.
There is thus a widely recognized need for, and it would be highly advantageous to have a method for the use of antioxidants in treatment of ischemic brain injuries, because antioxidants that would effectively cross the BBB and penetrate into the ischemic tissue mainly at its penumbra, may help to maintain the redox status of the neurons and decrease ROS-associated neuronal damage, thus reduce the neurological impairment and disability.
According to one aspect of the present invention there is provided a method of reducing oxidative stress in the brain of an organism having a blood brain barrier and suffering an ischemic brain injury, the method comprising the step of administering a compound to the organism, the compound having (a) a combination of molecular weight and membrane miscibility properties for permitting the compound to cross the blood brain barrier of the organism; (b) a readily oxidizable chemical group for exerting antioxidation properties; and (c) a chemical make-up for permitting the compound or its intracellular derivative to accumulate within the cytoplasm of cells.
According to another aspect of the present invention there is provided a method of therapeutically or prophylactically treating an individual against ischemic brain injury, the method comprising the step of administering to the individual a pharmaceutical composition to the individual, the pharmaceutical composition including a pharmaceutically acceptable carrier and a therapeutically or prophylactically effective amount of an antioxidant compound, the antioxidant compound having (a) a combination of molecular weight and membrane miscibility properties for permitting the compound to cross the blood brain barrier of the individual; (b) a readily oxidizable chemical group for exerting antioxidation properties; and (c) a chemical make-up for permitting the compound or its intracellular derivative to accumulate within brain cells of the individual.
According to yet another aspect of the present invention there is provided a pharmaceutical composition for therapeutically or prophylactically treating an individual against ischemic brain injury, the composition comprising a pharmaceutically acceptable carrier and, as an active ingredient, a therapeutically or prophylactically effective amount of an antioxidant compound, the compound having (a) a combination of molecular weight and membrane miscibility properties for permitting the compound to cross the blood brain barrier of the individual; (b) a readily oxidizable chemical group for exerting antioxidation properties; and (c) a chemical make-up for permitting the compound or its intracellular derivative to accumulate within brain cells of the individual.
According to further features in preferred embodiments of the invention described below, the compound is selected from the group consisting of N-acetyl cysteine ethyl ester (compound A), xcex2,xcex2-dimethyl cysteine ethyl ester (compound B), N-acetyl-xcex2,xcex2-dimethyl cysteine (compound C), Glutathione ethyl ester (compound D), N-acetyl glutathione ethyl ester (compound E), N-acetyl glutathione (compound F), N-acetyl xcex1-glutamyl ethyl ester cysteinyl glycyl ethyl ester (compound G) N-acetyl xcex1-glutamyl ethyl ester cysteinyl glycyl (compound H), N-acetyl glutathione amide (compound I), N-acetyl cysteine amide (compound J), N-acetyl xcex2,xcex2 dimethyl cysteine amide (compound K) and N-acetyl cysteine glycine amide.
According to still further features in the described preferred embodiments the ischemic brain injury is a result of a stroke or a head trauma.
According to still further features in the described preferred embodiments the pharmaceutically acceptable carrier is selected from the group consisting of a thickener, a carrier, a buffer, a diluent, a surface active agent and a preservatives.
According to still further features in the described preferred embodiments the administration is peripheral.
According to still further features in the described preferred embodiments the peripheral administration is selected from the group consisting of topical administration, oral administration, administration by inhalation, and parenteral administration.
According to still further features in the described preferred embodiments the readily oxidizable chemical group is a sulfhydril group.
According to still further features in the described preferred embodiments the chemical make-up is selected having an ester moiety which is removable by hydrolysis imposed by intracellular esterases.
According to still further features in the described preferred embodiments the administration is peripheral.
According to still further features in the described preferred embodiments the ester moiety is selected from the group consisting of alkyl ester and aryl ester.
According to still further features in the described preferred embodiments the alkyl and aryl esters are selected from the group consisting of methyl ester, ethyl ester, hydroxyethyl ester, t-butyl ester, cholesteryl ester, isopropyl ester and glyceryl ester.
According to still further features in the described preferred embodiments the organism is a human being.
The present invention successfully addresses the shortcomings of the presently known configurations by providing a novel method and pharmaceutical composition for the therapeutic or prophylactic treatment of ischemic brain injuries, such as stroke and head trauma.